Antonio Starcevic

ClustScan: an integrated program package for the semi-automatic annotation of modular biosynthetic gene clusters and in silico prediction of novel chemical structures

The program package ‘ClustScan’ (Cluster Scanner) is designed for rapid, semi-automatic, annotation of DNA sequences encoding modular biosynthetic enzymes including polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS) and hybrid (PKS/NRPS) enzymes. The program displays the predicted chemical structures of products as well as allowing export of the structures in a standard format for analyses with other programs. Recent advances in understanding of enzyme function are incorporated to make knowledge-based predictions about the stereochemistry of products. The program structure allows easy incorporation of additional knowledge about domain specificities and function. The results of analyses are presented to the user in a graphical interface, which also allows easy editing of the predictions to incorporate user experience. The versatility of this program package has been demonstrated by annotating biochemical pathways in microbial, invertebrate animal and metagenomic datasets. The speed and convenience of the package allows the annotation of all PKS and NRPS clusters in a complete Actinobacteria genome in 2–3 man hours. The open architecture of ClustScan allows easy integration with other programs, facilitating further analyses of results, which is useful for a broad range of researchers in the chemical and biological sciences.

Enzymes of the shikimic acid pathway encoded in the genome of a basal metazoan, Nematostella vectensis, have microbial origins.

The shikimic acid pathway is responsible for the biosynthesis of many aromatic compounds by a broad range of organisms, including bacteria, fungi, plants, and some protozoans. Animals are considered to lack this pathway, as evinced by their dietary requirement for shikimate-derived aromatic amino acids. We challenge the universality of this traditional view in this report of genes encoding enzymes for the shikimate pathway in an animal, the starlet sea anemone Nematostella vectensis. Molecular evidence establishes horizontal transfer of ancestral genes of the shikimic acid pathway into the N. vectensis genome from both bacterial and eukaryotic (dinoflagellate) donors. Bioinformatic analysis also reveals four genes that are closely related to those of Tenacibaculum sp. MED152, raising speculation for the existence of a previously unsuspected bacterial symbiont. Indeed, the genome of the holobiont (i.e., the entity consisting of the host and its symbionts) comprises a high content of Tenacibaculum-like gene orthologs, including a 16S rRNA sequence that establishes the phylogenetic position of this associate to be within the family Flavobacteriaceae. These results provide a complementary view for the biogenesis of shikimate-related metabolites in marine Cnidaria as a “shared metabolic adaptation” between the partners.

Spontaneity in the patellamide biosynthetic pathway.

Post-translationally modified ribosomal peptides are unusual natural products and many have potent biological activity. The biosynthetic processes involved in their formation have been delineated for some, but the patellamides represent a unique group of these metabolites with a combination of a macrocycle, small heterocycles and d-stereocentres. The genes encoding for the patellamides show very low homology to known biosynthetic genes and there appear to be no explicit genes for the macrocyclisation and epimerisation steps. Using a combination of literature data and large-scale molecular dynamics calculations with explicit solvent, we propose that the macrocyclisation and epimerisation steps are spontaneous and interdependent and a feature of the structure of the linear peptide. Our study suggests the steps in the biosynthetic route are heterocyclisation, macrocyclisation, followed by epimerisation and finally dehydrogenation. This study is presented as testable hypothesis based on literature and theoretical data to be verified by future detailed experimental investigations.

A profile of an endosymbiont-enriched fraction of the coral Stylophora pistillata reveals proteins relevant to microbial-host interactions

This study examines the response of Symbiodinium sp. endosymbionts from the coral Stylophora pistillata to moderate levels of thermal “bleaching” stress, with and without trace metal limitation. Using quantitative high throughput proteomics, we identified 8098 MS/MS events relating to individual peptides from the endosymbiont-enriched fraction, including 109 peptides meeting stringent criteria for quantification, of which only 26 showed significant change in our experimental treatments; 12 of 26 increased expression in response to thermal stress with little difference affected by iron limitation. Surprisingly, there were no significant increases in antioxidant or heat stress proteins; those induced to higher expression were generally involved in protein biosynthesis. An outstanding exception was a massive 114-fold increase of a viral replication protein indicating that thermal stress may substantially increase viral load and thereby contribute to the etiology of coral bleaching and disease. In the absence of a sequenced genome for Symbiodinium or other photosymbiotic dinoflagellate, this proteome reveals a plethora of proteins potentially involved in microbial-host interactions. This includes photosystem proteins, DNA repair enzymes, antioxidant enzymes, metabolic redox enzymes, heat shock proteins, globin hemoproteins, proteins of nitrogen metabolism, and a wide range of viral proteins associated with these endosymbiont-enriched samples. Also present were 21 unusual peptide/protein toxins thought to originate from either microbial consorts or from contamination by coral nematocysts. Of particular interest are the proteins of apoptosis, vesicular transport, and endo/exocytosis, which are discussed in context of the cellular processes of coral bleaching. Notably, the protein complement provides evidence that, rather than being expelled by the host, stressed endosymbionts may mediate their own departure.

KEGG orthology-based annotation of the predicted proteome of Acropora digitifera: ZoophyteBase - an open access and searchable database of a coral genome

BackgroundContemporary coral reef research has firmly established that a genomic approach is urgently needed to better understand the effects of anthropogenic environmental stress and global climate change on coral holobiont interactions. Here we present KEGG orthology-based annotation of the complete genome sequence of the scleractinian coral Acropora digitifera and provide the first comprehensive view of the genome of a reef-building coral by applying advanced bioinformatics.DescriptionSequences from the KEGG database of protein function were used to construct hidden Markov models. These models were used to search the predicted proteome of A. digitifera to establish complete genomic annotation. The annotated dataset is published in ZoophyteBase, an open access format with different options for searching the data. A particularly useful feature is the ability to use a Google-like search engine that links query words to protein attributes. We present features of the annotation that underpin the molecular structure of key processes of coral physiology that include (1) regulatory proteins of symbiosis, (2) planula and early developmental proteins, (3) neural messengers, receptors and sensory proteins, (4) calcification and Ca2+-signalling proteins, (5) plant-derived proteins, (6) proteins of nitrogen metabolism, (7) DNA repair proteins, (8) stress response proteins, (9) antioxidant and redox-protective proteins, (10) proteins of cellular apoptosis, (11) microbial symbioses and pathogenicity proteins, (12) proteins of viral pathogenicity, (13) toxins and venom, (14) proteins of the chemical defensome and (15) coral epigenetics.ConclusionsWe advocate that providing annotation in an open-access searchable database available to the public domain will give an unprecedented foundation to interrogate the fundamental molecular structure and interactions of coral symbiosis and allow critical questions to be addressed at the genomic level based on combined aspects of evolutionary, developmental, metabolic, and environmental perspectives.

Draft Genome Sequence of Streptomyces rapamycinicus Strain NRRL 5491, the Producer of the Immunosuppressant Rapamycin

ABSTRACT Streptomyces rapamycinicus strain NRRL 5491 produces the important drug rapamycin. It has a large genome of 12.7 Mb, of which over 3 Mb consists of 48 secondary metabolite biosynthesis clusters.

Gene Expression in the Scleractinian Acropora microphthalma Exposed to High Solar Irradiance Reveals Elements of Photoprotection and Coral Bleaching

Background The success of tropical reef-building corals depends on the metabolic co-operation between the animal host and the photosynthetic performance of endosymbiotic algae residing within its cells. To examine the molecular response of the coral Acropora microphthalma to high levels of solar irradiance, a cDNA library was constructed by PCR-based suppression subtractive hybridisation (PCR-SSH) from mRNA obtained by transplantation of a colony from a depth of 12.7 m to near-surface solar irradiance, during which the coral became noticeably paler from loss of endosymbionts in sun-exposed tissues. Methodology/Principal Findings A novel approach to sequence annotation of the cDNA library gave genetic evidence for a hypothetical biosynthetic pathway branching from the shikimic acid pathway that leads to the formation of 4-deoxygadusol. This metabolite is a potent antioxidant and expected precursor of the UV-protective mycosporine-like amino acids (MAAs), which serve as sunscreens in coral phototrophic symbiosis. Empirical PCR based evidence further upholds the contention that the biosynthesis of these MAA sunscreens is a ‘shared metabolic adaptation’ between the symbiotic partners. Additionally, gene expression induced by enhanced solar irradiance reveals a cellular mechanism of light-induced coral bleaching that invokes a Ca2+-binding synaptotagmin-like regulator of SNARE protein assembly of phagosomal exocytosis, whereby algal partners are lost from the symbiosis. Conclusions/Significance Bioinformatics analyses of DNA sequences obtained by differential gene expression of a coral exposed to high solar irradiance has revealed the identification of putative genes encoding key steps of the MAA biosynthetic pathway. Revealed also by this treatment are genes that implicate exocytosis as a cellular process contributing to a breakdown in the metabolically essential partnership between the coral host and endosymbiotic algae, which manifests as coral bleaching.

Predicting the nature and timing of epimerisation on a modular polyketide synthase

Modular polyketide synthases (PKSs) are multifunctional enzyme complexes that catalyse the synthesis of a structurally diverse group of secondary metabolites – the polyketides. Many polyketides are produced by Streptomyces and related filamentous bacteria ; some are of commercial significance including the antibiotics erythromycin A and rifamycin, the immunosuppressive macrolide rapamycin and the antitumour compound mithramycin. Since many of these compounds are high value biopharmaceuticals there has been intense interest over recent years in studying the chemistry and molecular biology of biosynthesis on these megasynthases. The modular PKS leading to the formation of the erythromycin polyketide backbone (6-deoxyerythronolide B synthase or DEBS) in Saccharopolyspora erythraea is one of the best studied, especially regarding stereochemistry. Polyketide biosynthesis proceeds via a Claisen condensation between simple carboxylic acids with inversion of configuration to give a (2R) methyl centre. However, the methyl stereochemistry present in the erythromycin polyketide backbone, namely (2R), (4R), (6S), (8R), (10R) and (12S), implies that two epimerisation steps occur. We used a bioinformatics approach to identify a putative epimerase function in the relevant ketoreduction (KR) domains. We postulate that C2 epimerization and C3 ketoreduction are coordinated processes and that the KR domains within modular polyketide synthases should be referred to as isomeroreductases to reflect their bifunctional nature.

Bacterial diversity of polluted surface sediments in the northern Adriatic Sea

Samples were collected from sea sediments at seven sites in the northern Adriatic Sea that included six sites next to industrial complexes and one from a tourist site (recreational beach). The samples were assayed for alkanes and polycyclic aromatic hydrocarbons. The composition of the hydrocarbon samples suggested that industrial pollution was present in most cases. A sample from one site was also grown aerobically under crude oil enrichment in order to evaluate the response of indigenous bacterial populations to crude oil exposure. Analysis of 16S rRNA gene sequences showed varying microbial biodiversity depending on the level of pollution--ranging from low (200 detected genera) to high (1000+ genera) biodiversity, with lowest biodiversity observed in polluted samples. This indicated that there was considerable biodiversity in all sediment samples but it was severely restricted after exposure to crude oil selection pressure. Phylogenetic analysis of putative alkB genes showed high evolutionary diversity of the enzymes in the samples and suggested great potential for bioremediation and bioprospecting. The first systematic analysis of bacterial communities from sediments of the northern Adriatic Sea is presented, and it will provide a baseline assessment that may serve as a reference point for ecosystem changes and hydrocarbon degrading potential--a potential that could soon gain importance due to plans for oil exploitation in the area.

Evolutionary concepts in natural products discovery: what actinomycetes have taught us

Actinomycetes are a very important source of natural products for the pharmaceutical industry and other applications. Most of the strains belong to Streptomyces or related genera, partly because they are particularly amenable to growth in the laboratory and industrial fermenters. It is unlikely that chemical synthesis can fulfil the needs of the pharmaceutical industry for novel compounds so there is a continuing need to find novel natural products. An evolutionary perspective can help this process in several ways. Genome mining attempts to identify secondary metabolite biosynthetic clusters in DNA sequences, which are likely to produce interesting chemical entities. There are often technical problems in assembling the DNA sequences of large modular clusters in genome and metagenome projects, which can be overcome partially using information about the evolution of the domain sequences. Understanding the evolutionary mechanisms of modular clusters should allow simulation of evolutionary pathways in the laboratory to generate novel compounds.